Recombinant DNA technology is a core subject for B. Pharm students, covering the principles and practical steps used to manipulate genes for therapeutic protein production, vaccine design, and drug development. This introduction reviews key concepts such as restriction enzymes, DNA ligase, vectors (plasmids, bacteriophages, BACs), cloning strategies, transformation methods, selectable markers, expression systems, and essential techniques like PCR, gel electrophoresis, and sequencing. Emphasis is placed on experimental workflow: DNA isolation, fragmentation, ligation, host introduction, and screening. Understanding these processes enables rational design and troubleshooting of molecular cloning experiments in pharmaceutical biotechnology. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the primary role of restriction endonucleases in recombinant DNA technology?
- To ligate DNA fragments together
- To cleave DNA at specific sequences
- To amplify DNA exponentially
- To transport DNA into host cells
Correct Answer: To cleave DNA at specific sequences
Q2. Which enzyme catalyzes the formation of phosphodiester bonds between DNA fragments during cloning?
- Restriction endonuclease
- DNA polymerase
- DNA ligase
- Reverse transcriptase
Correct Answer: DNA ligase
Q3. What feature of a plasmid vector ensures it is replicated inside a bacterial host?
- Multiple cloning site (MCS)
- Origin of replication (ori)
- Selectable marker gene
- Promoter for transcription
Correct Answer: Origin of replication (ori)
Q4. Which cloning vector is most suitable for carrying very large DNA fragments (hundreds of kilobases)?
- Plasmid
- Bacteriophage lambda
- BAC (Bacterial Artificial Chromosome)
- Cosmid
Correct Answer: BAC (Bacterial Artificial Chromosome)
Q5. In blue-white screening, what property does a white colony indicate?
- Colony contains plasmid without insert
- Insert disrupts lacZ gene, indicating successful cloning
- Plasmid was lost during transformation
- Cells are non-competent
Correct Answer: Insert disrupts lacZ gene, indicating successful cloning
Q6. When creating cDNA from mRNA, which enzyme is required to synthesize the first-strand DNA?
- DNA ligase
- Reverse transcriptase
- Taq DNA polymerase
- RNase H
Correct Answer: Reverse transcriptase
Q7. What is the main advantage of using a high-copy-number plasmid for cloning a gene for protein expression?
- Lower metabolic burden on host
- Higher yield of plasmid DNA and potentially higher protein expression
- Increased genomic integration frequency
- More stable maintenance without selection
Correct Answer: Higher yield of plasmid DNA and potentially higher protein expression
Q8. Which method is commonly used to introduce recombinant plasmid DNA into competent bacterial cells by creating temporary pores in the membrane?
- Gel electrophoresis
- Polymerase chain reaction (PCR)
- Electroporation
- Restriction digestion
Correct Answer: Electroporation
Q9. What does an expression vector contain that a basic cloning vector may lack?
- Multiple cloning site (MCS)
- Selectable antibiotic resistance gene
- A strong promoter and translation initiation signals for gene expression
- Origin of replication
Correct Answer: A strong promoter and translation initiation signals for gene expression
Q10. Which technique is used to verify the size of DNA fragments after restriction digestion?
- SDS-PAGE
- Agarose gel electrophoresis
- Mass spectrometry
- ELISA
Correct Answer: Agarose gel electrophoresis
Q11. What is the purpose of a selectable marker gene on a plasmid?
- To enable visual identification of colonies by color changes only
- To allow only transformed cells to grow under selective conditions
- To amplify the inserted gene
- To induce plasmid integration into host genome
Correct Answer: To allow only transformed cells to grow under selective conditions
Q12. Which type of end produced by many restriction enzymes facilitates directional cloning due to complementary overhangs?
- Blunt ends
- Sticky (cohesive) ends
- Triple-stranded ends
- Phosphorylated blunt ends
Correct Answer: Sticky (cohesive) ends
Q13. During subcloning, what is the usual next step after ligation of an insert into a vector?
- Transformation into competent host cells
- Direct sequencing without host propagation
- Transcription in vitro
- Protein purification
Correct Answer: Transformation into competent host cells
Q14. Which PCR component is essential for defining the start and end points of the amplified DNA fragment?
- dNTPs
- Taq polymerase
- Primers
- Template RNA
Correct Answer: Primers
Q15. What is a multiple cloning site (MCS) in a plasmid vector?
- A region encoding antibiotic resistance
- A stretch of several unique restriction sites facilitating insertion of DNA
- A promoter for high-level expression
- An origin of replication
Correct Answer: A stretch of several unique restriction sites facilitating insertion of DNA
Q16. Which screening method uses a labeled probe to detect specific DNA sequences in cloned colonies or libraries?
- Blue-white screening
- Colony PCR
- Hybridization using radioactive or fluorescent probes (colony blot hybridization)
- Restriction fragment length polymorphism (RFLP)
Correct Answer: Hybridization using radioactive or fluorescent probes (colony blot hybridization)
Q17. When constructing a cDNA library, what does the library represent?
- All genomic DNA sequences including introns
- The set of expressed genes (mRNA-derived) in a particular tissue or condition
- Only promoter regions of genes
- A collection of random chromosomal fragments
Correct Answer: The set of expressed genes (mRNA-derived) in a particular tissue or condition
Q18. Why is it important to dephosphorylate a vector after restriction digestion before ligation with an insert?
- To remove the origin of replication
- To prevent vector self-ligation and reduce background colonies
- To increase the size of the vector
- To make sticky ends blunt
Correct Answer: To prevent vector self-ligation and reduce background colonies
Q19. Which feature distinguishes a shuttle vector from a standard plasmid?
- Contains multiple promoters for prokaryotes only
- Can replicate in two different host species (e.g., E. coli and yeast)
- Has no selectable marker
- Is always high-copy-number
Correct Answer: Can replicate in two different host species (e.g., E. coli and yeast)
Q20. What role does RNase H play during cDNA synthesis?
- Degrades RNA in RNA–DNA hybrids to allow second-strand synthesis
- Synthesizes the first cDNA strand
- Ligates cDNA fragments together
- Removes primers used in PCR
Correct Answer: Degrades RNA in RNA–DNA hybrids to allow second-strand synthesis
Q21. Which sequencing method revolutionized verification of cloned DNA by reading nucleotide sequences directly?
- Sanger chain-termination sequencing
- Southern blotting
- Northern blotting
- Western blotting
Correct Answer: Sanger chain-termination sequencing
Q22. In recombinant protein production, why is codon optimization sometimes performed when expressing a eukaryotic gene in bacteria?
- To change the protein’s amino acid sequence
- To improve mRNA stability and translation efficiency in the bacterial host
- To remove the start codon
- To add introns for better expression
Correct Answer: To improve mRNA stability and translation efficiency in the bacterial host
Q23. What is the purpose of using a reporter gene (e.g., GFP or lacZ) in cloning and expression studies?
- To increase plasmid copy number
- To monitor expression visually or by assay
- To provide antibiotic resistance
- To induce genomic integration
Correct Answer: To monitor expression visually or by assay
Q24. Which safety concern is most relevant when performing recombinant DNA experiments in a teaching lab?
- Risk of electrical shock from gel boxes only
- Potential creation of harmful organisms and horizontal transfer of antibiotic resistance
- Likelihood of plasmid spontaneously combusting
- Risk of protein overproduction causing explosions
Correct Answer: Potential creation of harmful organisms and horizontal transfer of antibiotic resistance
Q25. What is the key difference between genomic DNA libraries and cDNA libraries?
- Genomic libraries contain only expressed sequences; cDNA libraries contain introns
- Genomic libraries include entire genomic sequences with introns and regulatory regions; cDNA libraries contain exon-only sequences derived from mRNA
- They are identical in content but use different vectors
- cDNA libraries are created from genomic DNA using restriction enzymes
Correct Answer: Genomic libraries include entire genomic sequences with introns and regulatory regions; cDNA libraries contain exon-only sequences derived from mRNA
Q26. Which practice helps reduce false positives when screening for recombinant clones?
- Omitting antibiotic selection
- Using negative controls and multiple screening methods (e.g., colony PCR plus restriction mapping)
- Using lower incubation temperatures for growth only
- Increasing vector concentration without dephosphorylation
Correct Answer: Using negative controls and multiple screening methods (e.g., colony PCR plus restriction mapping)
Q27. What is an operon commonly used for in bacterial expression systems?
- To provide eukaryotic splicing signals
- To coordinate transcription of linked genes under a single promoter
- To encode antibiotic resistance only
- To methylate foreign DNA
Correct Answer: To coordinate transcription of linked genes under a single promoter
Q28. How does electroporation differ from chemical transformation using CaCl2 in terms of efficiency and mechanism?
- Electroporation uses heat shock, CaCl2 uses electric field; electroporation is less efficient
- Electroporation applies an electric pulse to create transient pores and is generally more efficient than CaCl2 chemical competence
- Both use the same mechanism and have identical efficiencies
- CaCl2 requires linear DNA, electroporation requires circular DNA
Correct Answer: Electroporation applies an electric pulse to create transient pores and is generally more efficient than CaCl2 chemical competence
Q29. In cloning workflows, why is sequencing of the inserted DNA considered essential after screening?
- Sequencing is not necessary if blue-white screening worked
- To confirm correct insert identity, orientation, and absence of PCR-induced mutations
- To determine protein tertiary structure
- To increase plasmid copy number
Correct Answer: To confirm correct insert identity, orientation, and absence of PCR-induced mutations
Q30. Which modern genome-editing tool can be combined with recombinant DNA methods to create targeted gene modifications in pharmaceutical research?
- Sanger sequencing
- CRISPR-Cas systems
- Southern blotting
- Blue-white screening
Correct Answer: CRISPR-Cas systems
